Although examples of protein immobilization may be found dating to the early 1920's (Nelson, J. M. and E. I. Hitchcocks, J. Amer. Chem. Soc. 46:1956 [1921]), intense interest in the phenomenon is evident from the last quarter of a century. Immobilization strategies can be grouped into four general classes: (1) entrapment (2) cross-linking (3) covalent binding and (4) adsorption.
Entrapment strategies are generally based upon occlusion within cross-linked gels or encapsulation with hollow fibers, liposomes, microcapsules and the like. Cross-linking involves modification of the enzyme by the addition of so-called bi-or multifunctional cross-linking reagents often following adsorption or encapsulation. Covalent-linking, the most widely investigated strategy, involves the covalent binding of the enzyme to a support by means of functional groups which are nonessential for the biological activity of the enzyme. Adsorption is achieved by simply contacting the enzyme with an adsorbent and allowing the immobilization to result from the interaction of the relatively weak binding forces between the enzyme and the adsorbent.
It is to this latter strategy of immobilization that this invention most directly relates; therefore, certain principles of adsorption will be developed more fully below. Comprehensive reviews of entrapment, cross-linking and covalent binding exist (See for example: Weetal, H. H., ed. "Immobilized Enzymes, Antigens, Antibodies and Peptides" M. Dekker, New York, [1975], or Zaborsky O. R. "Immobilized Enzymes" CRC Press, Cleveland, [1973]).
Early investigation concerning adsorption indicated that in certain cases the adsorption could lead to partial or total inactivation of the enzyme. It is therefore appreciated that a suitable adsorbent for the practice of this invention is one which possesses a relatively high affinity for the enzyme, yet causes minimal inactivation.
Adsorption immobilization to such supports as alumina, bentonite, calcium carbonate, cellulose, collagen, ion exchange resins, kalinite, Sephadex, silica gel and titanium-coated stainless steel is known.
Although, as disclosed below, this invention is adaptable to a wide range of adsorbed enzyme systems, it is particularly suited for the beneficiation of processes employing immobilized glucose isomerase.
Most fructose is commercially produced by isomerizing dextrose (from starch) to fructose in a reactor whereby dextrose solution is passed through a bed of immobilized glucose isomerase. The fructose content of effluent is typically held at a constant level. e.g., between 40-44%, by controlling the flow rate through the reactor. As the immobilized enzyme naturally decays due to thermal and chemical inactivation the flow rate is periodically reduced. When a flow rate is reached beyond which further reduction is impractical the immobilized enzyme is replaced with a new bed of immobilized enzyme. Because flow rate of a column during its lifetime may vary from, e.g., 50 GPM to 4 GPM, numerous columns must be in place to provide a nearly constant production rate.
Particularly useful systems for the immobilization of glucose isomerase have been described in U.S. Pat. Nos. 3,788,945, 3,909,354, 4,110,164, 4,168,250 and 4,355,117. U.S. Pat. No. 3,960,663 describes periodic addition of soluble glucose isomerase via the dextrose feed stream to an isomerization reactor containing immobilized glucose isomerase. The glucose isomerase is immobilized to a strong base anion-exchange resin which has been loaded to capacity with soluble glucose isomerase. As the enzyme decays, its adsorption properties are altered such that the inactive enzyme is sloughed off the support and appears in the eluent. The patent teaches to replace the leeched enzyme by contacting the support with fresh enzyme in an amount sufficient to totally re-charge the support. Since the spent enzyme is continually leeched from the adsorbent the eluent must be subjected to a further purification step in order to remove the contaminating enzyme.
The application of the subject invention leads to significant advantages over conventional reactions with respect to: (1) simplification of operations--the need to adjust reactor flows on a periodic bases would be eliminated; (2) lower capital investment--smaller reactors would be needed in favor of a few large reactors due to elimination of fluctuating flow rates; (3) improved production control--higher fructose levels could be achieved with no need for reduced flow rates by addition of more soluble enzyme to the reactor; and, (4) reduced product-refining costs--because very slow flow rates and hence long residence times would be eliminated, production of color and off-flavors would be lessened.